SECRETION OF ACTH, LPH AND β-ENDORPHIN FROM HUMAN PITUITARY TUMOURS IN VITRO

Basal and stimulated secretion of immunoreactive ACTH, LPH and β-endorphin from four human pituitary tumours has been studied in vitro using a superfused, isolated cell system. Chromatography of cell secretions under acid-dissociating conditions demonstrated that the human tumour cells released immunoreactive peptides with the elution profiles of α_h (1–39) ACTH, β_h-LPH, γ_h-LPH and β_h-endorphin confirming that β_h-endorphin is secreted by human pituitary tumour cells and is not formed by enzymic cleavage from β_h-LPH in blood. No α- or β_h-MSH, nor any higher molecular weight forms of ACTH or LPH were detected. The cells from all four tumours responded to stimulation with rat stalk-median eminence extract (SME) and synthetic AVP with a concomitant release of ACTH, β-LPH, γ-LPH and γ-endorphin. In contrast to the isolated rat anterior pituitary cells, the pattern of responses to SME and AVP were indistinguishable and the release provoked by rat SME could be accounted for virtually entirely by its vasopressin content. No stimulation of release was observed when the cells were exposed to a variety of biogenic amines. Addition of hydrocortisone to the perfusion buffer of two tumours resulted in a slow inhibition of both basal and stimulated ACTH and LPH release. These data demonstrate that human pituitary tumour tissue from patients with Cushing's disease and Nelson's syndrome can be studied in vitro and that such studies may contribute to a greater understanding of the aetiology of these diseases.     

Immunological characterization of β-lipotropin fragments (endorphin, β-MSH,and N-fragment) from fish pituitaries

Radioimmunoassay (RIA) for each of the three segments of the β-lipotropin molecule (N-fragment, β-MSH, and β-endorphin) are described. The pituitaries of one elasmobranch and two teleost species contain the three segments in the following order of abundance: β-endorphin > β-MSH > N-fragment. By gel filtration we have shown that the majority of the β-MSH and β-endorphin immunoreactivity is accounted for by the free peptides, not by larger prohormones. Only very small amounts of immunoreactive material the size of β- or γ-LPH were found.     

Deciphering Posttranslational Processing Events in the Pituitary of a Neopterygian Fish: Cloning of a Gar Proopiomelanocortin cDNA

A cDNA that codes for the polypeptide hormone precursor proopiomelanocortin (POMC) was cloned and sequenced from a gar (Lepisosteus osseus) pituitary cDNA library. The gar POMC cDNA is 1237 bp and contains a 780-bp open reading frame. The deduced amino acid sequence for gar POMC is 259 amino acids in length. The general organization of gar POMC is very similar to that of other gnathostome POMC sequences. The β-endorphin sequence had 91% sequence identity with sockeye A β-endorphin and 71% sequence identity withXenopus laevisβ-endorphin. Three melanocyte-stimulating hormone (MSH) core sequences [HFR(W)] were detected. The gar α-MSH sequence was identical to the α-MSH sequence in rat POMC. The gar β-MSH sequence had 77% sequence identity with salmonid forms of β-MSH and 53% sequence identity with tetrapod forms of β-MSH. The γ-MSH region of gar POMC only had 26% primary sequence identity with tetrapod γ-MSH sequences. Gar γ-MSH had an incomplete MSH core sequence (HRF), an apparent internal deletion of five amino acids, and lacked flanking paired basic amino acids essential for proteolytic cleavage. The apparent degenerate nature of gar γ-MSH is discussed in light of the absence of this sequence in salmonid fish.     

The evolution of vertebrate corticotrophin and melanocyte stimulating hormone.

ACTH and β-LPH appear to be synthesised in both the pars distalis and the pars intermedia. In the pars distalis they are secreted intact. In the pars intermedia ACTH acts as the precursor of α-MSH and CLIP, and β-LPH acts as the precursor for γ-LPH and β-MSH. α-MSH, CLIP and β-MSH do not exist in significant amounts in the human pituitary. α-MSH, β-MSH, ACTH, and CLIP have been characterised in the elasmobranch pituitary. Chicken ACTH has been isolated.     

Role of proopiomelanocortin-derived peptides and their receptors in the osteoarticular system: from basic to translational research

Proopiomelanocortin (POMC)-derived peptides such as melanocortins and β-endorphin (β-ED) exert their pleiotropic effects via binding to melanocortin receptors (MCR) and opioid receptors (OR). There is now compelling evidence for the existence of a functional POMC system within the osteoarticular system. Accordingly, distinct cell types of the synovial tissue and bone have been identified to generate POMC-derived peptides like β-ED, ACTH, or α-MSH. MCR subtypes, especially MC1R, MC2R (the ACTH receptor), MC3R, and MC4R, but also the μ-OR and δ-OR, have been detected in various cells of the synovium, cartilage, and bone. The respective ligands of these POMC-derived peptide receptors mediate an increasing number of newly recognized biological effects in the osteoarticular system. These include bone mineralization and longitudinal growth, cell proliferation and differentiation, extracellular matrix synthesis, osteoprotection, and immunomodulation. Importantly, bone formation is also regulated by the central melanocortin system via a complex hormonal interplay with other organs and tissues involved in energy metabolism. Among the POMC-derived peptides examined in cell culture systems from osteoarticular tissue and in animal models of experimentally induced arthritis, α-MSH, ACTH, and MC3R-specific agonists appear to have the most promising antiinflammatory actions. The effects of these melanocortin peptides may be exploited in future for the treatment of patients with inflammatory and degenerative joint diseases.     

Immunocytochemical evidence for melanotropin- and vasopressin-like material in a cephalopod neurohemal organ

Antibodies against the peptide hormone α-melanotropin (α-MSH) and antibodies against arginine-vasopressin (AVP) recognized material in two different sorts of nerve endings of the “neurosecretory system of the vena cava” in the octopus. AVP-like immunoreactivity occurred together with neurophysin-like immunostaining in the same terminals. Among the tested antibodies against different portions of the proopiocortin molecule, including β-endorphin, corticotropin (ACTH), and the 16,000 MW fragment, only anti-α-MSH serum produced immunostaining. These observations may indicate that some of the smallest bioactive peptides of the vertebrate pituitary have persisted for long periods in evolution.     

On the potential role of proopiomelanocortin in skin physiology and pathology

Pro-opiomelanocortin (POMC) is the precursor of ACTH, α-MSH and β-endorphin, neuropeptides with multiple regulatory functions. Both the pituitary gland and peripheral tissues such as mammalian skin are capable of generating POMC-derived neuropeptides. Mammalian skin is also a target for POMC products; their possible roles in skin physiology and pathology are discussed in this communication.     

In vitro study of frog (Rana ridibunda pallas) neurointermediate lobe secretion by use of a simplified perifusion system: III. Effect of neuropeptides on α-MSH secretion

It has been previously demonstrated that thyrotropin-releasing hormone (TRH) stimulates in vitro the release of α-melanocyte-stimulating hormone (α-MSH) in frog. In the present study, the effects of various neuropeptides on spontaneous and/or TRH-induced α-MSH secretion were investigated, using a well-defined perifusion system technique. Vasoactive intestinal peptide, (VIP) a neurohormone which stimulates TRH target cells in mammals, was totally devoid of effect on frog melanotrophs although VIP-like material could be detected in neurointermediate lobe extracts. Somatostatin-like immunoreactive material was found in high concentrations in the frog neurointermediate lobe complex, but synthetic somatostatin (from 10^?10 to 10^?6M) did not modify the spontaneous release of α-MSH. At doses of 10^?8 and 10^?6M, synthetic somatostatin did not modify TRH-induced α-MSH secretion. Morphine (10^?5M) and opioid peptides (10^?10 to 10^?6M) had no effect on spontaneous α-MSH secretion. In addition, methionine enkephalin (10^?5M) did not modify the stimulatory effect of TRH on α-MSH secretion. From these results we conclude that, among the neuropeptides which modulate prolactin secretion in mammals, only TRH is involved in α-MSH secretion in the frog.     

Degree of inhibition of ACTH release by glucocorticoids in adrenalectomized rats

The inhibitory effects of corticosterone, dexamethasone and prednisolone on activity of the hypothalamus-pituitary-adrenal axis were investigated in adrenalectomized rats infused with glucocorticoids for 6 h. Infusion of 202 micrograms corticosterone did not inhibit the plasma ACTH concentration, but 504 micrograms corticosterone significantly suppressed plasma ACTH levels. Infusion of 20 micrograms dexamethasone suppressed markedly the plasma ACTH concentration. These data suggest that the degree of inhibition of dexamethasone on ACTH release is about 25 times greater than that of corticosterone. The CRF content of the hypothalamus was not decreased by the administration of 202 micrograms corticosterone over a 6-hour period, but it was significantly diminished by 504 micrograms corticosterone. Infusion of 504 micrograms dexamethasone did not decrease the hypothalamic CRF content; however, infusion of 5 mg dexamethasone effectively suppressed the hypothalamic CRF content. Infusion of 2.5 mg prednisolone did not either decrease the CRF content. These data suggest that the degree of inhibition of natural steroid at the hypothalamus level is stronger than that of synthetic steroids. In rats pretreated with a single injection of dexamethasone (25 micrograms/200 g body weight) 22 h prior to the experiments, continuous infusion of 318 micrograms of dexamethasone significantly suppressed the hypothalamic CRF content, whereas infusion of 504 micrograms of dexamethasone failed to decrease the hypothalamic CRF content in the rats not pretreated with dexamethasone. This finding suggests that a latent period after the injection of dexamethasone is needed for the appearance of the inhibitory action of synthetic steroids at the level of hypothalamus.     

CLINICAL EVALUATION OF A RADIOIMMUNOASSAY FOR β-MSH-RELATED PEPTIDES (LIPOTROPHINS) IN HUMAN PLASMA

A radioimmunoassay for β-MSH related peptides (‘β-MSH’) which measures βh- and γh-lipotrophin (LPH) and β-MSH, was used to assess LPH status and the stability of ‘β-MSH’ in blood and plasma.‘β-MSH’ concentrations correlated well with ACTH levels in paired snap-frozen plasma samples from normal subjects, from patients with pathologically elevated hormone levels (except those with chronic renal failure), and during acute stimulation and suppression tests (r= 0.913, P > 0.001). The mean ratio of ‘β-MSH’ and ACTH levels in most situations was approximately unity. In chronic renal failure,‘β-MSH’ concentrations were significantly higher than ACTH (mean ratio 4.1). Lesser degrees of dissociation of hormone levels were also found in patients with the ectopic ACTH syndrome (mean ratio 2.8). Basal ‘β-MSH’ levels were uniformly elevated in patients with Cushing's disease after bilateral adrenalectomy, Nelson's syndrome, ectopic ACTH syndrome, and Addison's disease and overlapped the normal range in patients with untreated Cushing's disease. There was complete separation of ‘β-MSH’ levels in ACTH-dependent and ACTH-independent (adrenal tumour) causes of Cushing's syndrome. Endogenous ‘β-MSH’ immunoactivity was more stable than ACTH in blood and plasma. There was no significant decrease of ‘β-MSH’ immunoactivity in samples kept at ambient temperature for 24 h or after four cycles of freezing and thawing plasma.‘β-MSH’ levels also did not change significantly in unhaemolysed blood samples transported at ambient temperature by first class mail. It is concluded that the ‘β-MSH’ assay provides at least as good discrimination as the ACTH assay in situations in which the latter is diagnostically valuable. Determination of the ratio of ‘β-MSH’ to ACTH levels may help to distinguish between untreated Cushing's disease and the ectopic ACTH syndrome. The stability of ‘β-MSH’ immunoactivity allows unhaemolysed blood and plasma samples to be transported at ambient temperature without loss of diagnostic value.     

Evaluation of posttranslational processing of proopiomelanocortin in the banded houndshark pituitary by combined cDNA cloning and mass spectrometry

Proopiomelanocortin (POMC) is cleaved into small peptides, such as adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormones (MSHs), and β-endorphin (β-END), by tissue-specific posttranslational processing in the corticotrophs of the pars distalis (PD) and melanotrophs of the neurointermediate lobe (NIL) of the pituitary. We examined the posttranslational processing of POMC in the pituitary of the banded houndshark Triakis scyllium by molecular cloning and subsequent mass spectrometric identification of the POMC-derived peptides in the pituitary extracts. One-fifth of the randomly selected clones from a Triakis pituitary cDNA library contained a cDNA encoding for POMC. Triakis prePOMC contained 4 MSHs and a single β-END, as has been observed in case of other cartilaginous fish POMCs. These predicted hormonal segments were flanked by basic amino acid residues, which are the cleavage sites for the processing enzymes, i.e., protein convertases. Mass spectrometry was performed using PD (including most parts of the rostral and proximal PD) and NIL extracts to detect mass values corresponding to the POMC-derived peptides. Consequently, ACTH, β-END, and the joining peptide (JP) were detected in the PD extract, while MSHs, processed β-END, and some other POMC-derived peptides were identified in the NIL extract; however, neither acetylated α-MSH nor acetylated β-END was detected in the latter. These tissue-specific POMC processing patterns are similar to those of the other vertebrate pituitaries; however, the absence of acetylated peptides suggests the lack of an acetylation system in the melanotrophs in the NIL of the Triakis pituitary.     

The effect of β-endorphin on basal and insulin-hypoglycaemia stimulated levels of hypothalamic-pituitary-adrenal axis hormones in normal human subjects

OBJECTIVE It has been demonstrated that β-endorphin reduces CRH production and hypoglycaemia-induced ACTH secretion in the rat. We aimed to determine whether supraphysiological levels of β-endorphin inhibit the ACTH and CRH response to insulin-induced hypoglycaemia in human subjects. DESIGN Plasma glucose, prolactin, cortisol, ACTH, CRH and AVP were measured at intervals over a 3-hour period. Intravenous β-endorphin 5 mg/50 ml or an equal volume of normal saline was infused between 30 and 90 minutes, with soluble insulin 0.15 units/kg administered i.v. at 60 minutes in a cross-over design. SUBJECTS Six healthy male volunteers aged 20–35 years. MEASUREMENTS Prolactin was measured by a fluoroimmunometric assay, ACTH, CRH and AVP by radioimmunoassay, and cortisol was measured by enzyme-linked immunosorbent assay. Haemodynamic measurements were recorded prior to each blood sample. Results are expressed as mean ± standard error of the mean. RESULTS β-Endorphin resulted in a significant decrease in baseline cortisol (P < 0.05) but not ACTH. Plasma glucose (P < 0.001) and CRH (P < 0.05) and PRL (P < 0.05) increased significantly during β-endorphin compared to normal saline. After insulin administration, glucose reached a similar nadir during β-endorphin and normal saline (2.1 ± 0.1 and 1.9 ± 0.15 mmol/l, respectively) but the fall in plasma glucose was delayed during β-endorphin (P < 0.01 by ANOVA). This resulted in a significantly altered time-course for the ACTH and cortisol responses (P < 0.05 for each), but no difference overall in the magnitude of the response. In contrast, neither the timing nor the magnitude of the CRH and AVP responses were affected. Prolactin also reached a similar peak value after the administration of insulin, while the haemodynamic responses to hypoglycaemia were not significantly altered during β-endorphin. CONCLUSIONS While β-endorphin has been shown to be inhibitory to basal ACTH and cortisol secretion in humans, we note a significant increase in plasma CRH in response to β-endorphin, which may be arising from a peripheral source. Intravenous β-endorphin increases plasma glucose and delays the onset of hypoglycaemia following insulin but does not result in significant inhibition of the ACTH and cortisol response. This may reflect the poor penetration of β-endorphin into the central nervous system, although a hypothalamic effect of β-endorphin is implied by the increased PRL. The significantly delayed time course in ACTH and cortisol secretion noted during β-endorphin is not explained by a later response of either CRH or AVP. Although peripheral levels of these hormones may be a relatively insensitive measure of hypothalamic function, an additional factor may influence ACTH release during hypoglycaemia.     

Melanotropin bioassays: In vitro and in vivo comparisons

A reflectance method was utilized to compare the in vitro responses in three species of frogs (Rana pipiens, R. berlandieri forrei, and R. catesbeiana) and a lizard (Anolis carolinensis) to α- and β-melanotropins (α- and β_p-MSH). The integumental chromatic response of the three ranid species was identical, in that α-MSH was about 2 times more potent than β_p-MSH. The melanotropins were equipotent in the lizard skin bioassay. A remarkable feature of the Anolis skin assay is that skins from this lizard can be utilized repeatedly many times in one day with an extremely high degree of precision. The reflectance method was also used to determine the in vivo potencies of α-MSH and β_p-MSH in the frog, R. pipiens. Surprisingly, the melanotropins were more active in the in vivo assay than in the in vitro bioassay. The darkening response of the frogs to α-MSH was reversed by 6 hr, but the response to β_p-MSH persisted for more than 8 hr. When α-MSH was incubated in frog serum, the melanotropic activity was almost totally abolished by 30 min, whereas the melanotropic activity of β_p-MSH was evident much longer (4 hr) in the presence of the serum. In light of the observation that the melanotropic activity of α-MSH is rapidly lost by incubation in frog serum, it is unclear why the hormone was more active as measured in vivo and why the darkening response in vivo persisted so long.     

Effects of adrenalectomy and dexamethasone administration on the level of prepro-corticotropin-releasing factor messenger ribonucleic acid (mRNA) in the hypothalamus and adrenocorticotropin/beta-lipotropin precursor mRNA in the pituitary in rats

RNA blot hybridization analysis with cloned rat CRF precursor (prepro-CRF) cDNA as a probe showed that prepro-CRF mRNA existed in rat hypothalamic and extrahypothalamic brain tissue, whereas it was undetectable in the pituitary and adrenal. To study the effect of glucocorticoid on the level of prepro-CRF mRNA in the hypothalmus and that of ACTH/beta-lipotropin (beta LPH) precursor mRNA in the pituitary, effects of adrenalectomy and dexamethasone administration were studied in rats. Adrenalectomy markedly raised mRNA coding for ACTH/beta LPH precursor in the anterior pituitary, but not in the neurointermediate pituitary lobe. Hypothalamic pre-pro-CRF mRNA increased only to 152% of the control value, 7 days after adrenalectomy. The administration of dexamethasone (200 micrograms/day for 7 days) started immediately after adrenalectomy lowered the ACTH/beta LPH precursor mRNA level in the anterior pituitary to 19% of the intact control value, whereas the level of prepro-CRF mRNA in the hypothalamus decreased only to 102%. These results suggest that glucocorticoids exert their feedback effect at the level of gene expression on both hypothalamic CRF neurons and pituitary corticotropes. Although the possibility that CRF neurons insensitive to glucocorticoid in the hypothalamus might blunt the change in the prepro-CRF mRNA could not be ruled out, it is also possible that the effect of glucocorticoids on the pituitary is dominant.     

A Radioimmunoassay for plasma corticotropin in frogs (Rana esculenta L.).

A radioimmunoassay technique has been developed for measuring frog plasma corticotropin (ACTH) without prior extraction. Using synthetic porcine ACTH as a reference standard, ¹³¹I-labeled synthetic human ACTH (sp act > 500 mCi/mg) as tracer and rabbit anti-porcine ACTH serum, the lower measurable value was estimated at about 4 pg ACTH. Only human and porcine ACTH, ^1–24ACTH, and frog pituitary ACTH reacted with the rabbit anti-porcine ACTH serum. No cross-reactivity has been found with synthetic ^{1–16,17–39}ACTH, αMSH, and bovine βMSH. Appearance of damaged ¹³¹I-h ACTH components after storage in plasma solutions was followed for 7 days. The conditions making it possible to reduce ACTH damage have been ascertained. The average plasma corticotropin level (±CI) was found to be 38.8 ± 7.8 pg/ml without any significant difference between males and females. These results suggest that frog ACTH secretion has much in common with mammalian secretions.     

β-Endorphin Antagonizes the Effects of α-MSH on Food Intake and Body Weight

Proopiomelanocortin (POMC) is posttranslationally processed to several peptides including α-MSH, a primary regulator of energy balance that inhibits food intake and stimulates energy expenditure. However, another POMC-derived peptide, β-endorphin (β-EP), has been shown to stimulate food intake. In this study we examined the effects of intracerebroventricular (icv) β-EP on food intake and its ability to antagonize the negative effects of α-MSH on energy balance in male rats. A single icv injection of β-EP stimulated food intake over a 2- to 6-h period during both the light and dark cycles. This effect was, however, not sustained with chronic icv β-EP infusion. In the next study, a subthreshold dose of β-EP was injected together with Nle(4), d-Phe(7) (NDP)-MSH after a 16-h fast, and the negative effects of NDP-MSH on refeeding and body weight gain were partially reversed. Finally, peptide interactions were studied in a chronic icv infusion model. Weight gain and food intake were significantly suppressed in the NDP-MSH group during the entire study. A subthreshold dose of β-EP antagonized these suppressive effects on food intake and weight gain for the first 3 d. However on d 4-7, β-EP no longer blocked these effects. Of note, the stimulatory effect of β-EP on feeding and its ability to antagonize MSH were specific for β-EP(1-31) and were not observed with β-EP(1-27). This study highlights the importance of understanding how the balance between α-MSH and β-EP is maintained and the potential role of differential POMC processing in regulating energy balance.     

Coexistence of alpha-melanocyte-stimulating hormone and adrenocorticotrophin in all cells containing either of the two hormones in the duck pituitary

Since birds lack the pars intermedia of the pituitary gland, the presence of ACTH-, α-MSH-, and β-MSH-containing cells in the pars distalis of the duck was investigated. By using the peroxidase-anti-peroxidase unlabeled antibody enzyme technique there were found cells reacting positively with antibodies against ACTH^1–24, ACTH^17–39, and α-MSH. Positive reactions could not be obtained with anti-bovine β-MSH serum. The coexistence of α-MSH and ACTH in all cells containing either of the two hormones could be established.     

Effects of dexamethasone on central and peripheral ACTH systems in the rat

To investigate the simultaneous effects of dexamethasone on peripheral and central adrenocorticotropic hormone (ACTH) systems, rats were treated with dexamethasone or saline for 4 days. Pituitary, plasma, hypothalamus and cerebrospinal fluid (CSF) were then collected and analyzed for ACTH immunoreactivity. Additionally, hypothalamic tissue extracts were analyzed for corticotropin-releasing hormone (CRH) immunoreactivity. Dexamethasone significantly lowered peripheral levels of ACTH as measured in pituitary and plasma. Hypothalamic ACTH content significantly increased while CSF ACTH significantly decreased with dexamethasone treatment. Hypothalamic CRH concentrations showed a small but statistically insignificant decrease. These results suggest that prolonged exposure to dexamethasone affects central as well as peripheral ACTH activity, corroborate our previous findings in rhesus monkeys of decreased CSF ACTH in response to prolonged dexamethasone treatment, suggest that dexamethasone may inhibit the release of ACTH from hypothalamic neurons into the CSF, and provide evidence that the effect of dexamethasone on pituitary ACTH content is of greater magnitude than its effect on hypothalamic CRH.     

A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. II. Hypothalamus-pituitary cell-adrenal cell superfusion.

Basal and stimulated CRF release by hypothalamic blocks was studied by coupling the effluent of superfused hypothalamus tissue to a joint pituitary cell-adrenal cell superfusion system and measuring corticosterone production. Log dose-response curves of the adrenal cells for ACTH and of the pituitary cell-adrenal cell system for CRF were linear over the ranges used. Ca++-independent basal CRF release by the hypothalamus could be blocked in vitro by 0.2 mug/ml dexamethasone in the medium, or in vivo by treating the hypothalamus donor rats with corticosterone, 1 mg/rat ip 30 min before decapitation. These treatments did not impair CRF release caused by Veratridine (5 x 10(-6)M or by electrical stimulation. Adrenalectomy increased only basal but not stimulated CRF release. These results indicate that glucocorticoids have a hypothalamic site of action.